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Vol. 9(21), pp.

1424-1430, 27 May, 2015


DOI: 10.5897/AJMR2015.7432
Article Number: 89FB65853286
ISSN 1996-0808
Copyright 2015
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJMR

African Journal of Microbiology Research

Full Length Research Paper

The effect of nano-TiO2 and plant extracts on microbial


strains isolated from Theban ancient Egyptian royal
tomb painting
Rafat KHALAPHALLAH1* and Abdou A.O. D. El-DERBY2
1

Microbiology Department, Faculty of Agriculture, South Valley University, Qena, Egypt.


Archaeology Conservation Department, Faculty of Archeology, South Valley University, Qena, Egypt.

Received 16 February, 2015; Accepted 13 April, 2015

Mural paintings in ancient Egyptian tombs in West Thebes have been suffering from several
deterioration factors and symptoms such as variations of temperatures and relative humidity, salts
efflorescence and crypto-florescence, crackling and bio-deterioration effects, which assimilate in
insects, algae, actinomycetes, etc. Other causing factors are bacteria and fungi, which accelerate
mechanical weathering, chemical changes and aesthetic deterioration, like the penetration of mycelium
below plaster layers, decomposition, disintegration, alterations and discoloration. These microorganisms can excrete organic and inorganic acids, alkaline compounds, chelating, enzymes
substances and pigments. Three fungi strains (Fusarium oxysporum, Rhizopus stolonifer and
Aspergillus flavus) and two bacteria strains (Staphylococcus warnei and Micrococcus luteus) were
isolated from the royal Theban tombs paintings (west Thebes, Luxor, Egypt). This work aimed to access
the presence of microorganisms and their effect on mural paintings deterioration; it also studies their
treatment methods, such as nanoparticles (TiO2 NPs) and Sesbania sesban and Ricinus communis plant
extract (PE). The applied doses of NPs and PE did not cause any observable alterations or color
changes to pigments and binding media (arabic gum) used in the paintings. TiO 2 NPs 160 ppm and 100
mg of plant extracts were the efficient concentration level in eliminating microbial growth. The causes of
the different efficacy of the treatments are observed, as well as the potential risks of recolonization by
viable cells left behind after treatment.
Key words: Bio-deterioration, Plant extracts, Wall painting, West Thebes tombs-Nanoparticles.

INTRODUCTION
Bio-deterioration can be defined as any undesirable
change in a material brought about by the vital activities
of organisms (Allsopp, 2011). Bacteria, actinomycetes
and fungi are constantly causing problems in the

conservation of mural paintings tombs because of their


biodeteriorative potential. The Thebes town is one of the
largest and famous archaeological sites in the world. It
lies about 900 km South of Cairo on the banks of the

*Corresponding author. E-mail: r.shipat@agr.svu.edu.eg.


Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0
International License

Khalaphallah and El-Derby

1425

1-

Figure 1. Plan of Luxor and west bank Luxor.

River Nile. The tradition of wall painting is very old, dating


back to prehistoric times (Garg et al., 2010). Royal tombs
in Thebes (west Bank, Modern Luxor) are as considered
one of the most important archaeological features in
Egypt and the world over. That is, where this
archaeological site is the most important period of
Egyptian history, tombs were constructed for the
Pharaohs and powerful nobles of the New Kingdom (the
Eighteenth to the Twentieth Dynasties of Ancient Egypt).
Wall painting generally classified as tempera or fresco
depending on the technique of execution, possesses a
layered structure consisting of support, ground and paint
layer (Garg et al., 2010). Wall painting supports the
growth of microorganism commonly involved in biodeterioration, contributing to the destruction of paint; their
deterioration constitutes a loss affecting a significant part
of the world's cultural heritage (Pepe et al., 2010). The
growth of fungi on wall paintings manifested most
commonly by discoloration or deterioration of the
surfaces and microbial popullation causes discoloration
of pigments, physical decay when fungal hyphae grow
either on or below the surfaces, cause chemical decay of
paintings through their metabolites and enzyme
production like gluconic, citric, oxalic, malic etc (Pepe et
al., 2010; Hideo, 2000; Dornieden et al., 2000; Garg et
al., 2010). Biodegradation and bio-deterioration is a
serious risk to cultural heritage, which needs the
application of effective and fast methods in order to
identify the microorganisms involved in this process and
to assess their biodegradation and bio-deterioration
ability (Pangallo et al., 2009). Their biological attack
occurs at favourable temperature and relative humidity
conditions for the development of microorganisms and
spores present on the substratum. Each colonizer agent
has different ways to compromise the structure in

function of the substrates (Nugari et al., 1993; Borrego et


al., 2010). Nanoparticles have antimicrobial effect such
as silver nano particles, copper nanoparticles, zinc oxide
nanoparticles and titanium oxide nano particles etc
(Stoimenov, 2002; Ip et al., 2006; Ren et al., 2009; Zhang
et al., 2007). In this study we used two methods to inhibit
the isolated microbial from the surface of wall painting:
the first involves using titanium dioxide nanoparticles with
a different level of concentration and the second method
entails using two tradition plant extracts- Sesbania
(Sesbania sesban) and Ricinus (Ricinus communis).
MATERIALS AND METHODS
Samples and site description
Samples for the study were taken from different parts of the royal
tombs such as Tausert and Setnkht, Seti ., Ramsis v., Ramsis vi.
(Figure 1) according to scientific and not destructive methods. The
biological samples were taken by sterile cotton swap and incubated
for 7 days; the isolated fungi were identified according to
morphological and spore structures (Barnett et al., 1972; Sun et al.,
1978).
Three isolates of fungi and two of bacteria were collected from
different tombs; 900 km Southeast of Cairo, during March 2014 by
using the sterile cotton swap method. Samples were taken from
paintings of yellow, red, black, blue color and stone surfaces in
investigated tombs (Figure 2). They were inoculated on nutrient
agar and potato dextrose agar, and incubated for a period of 5 days
at 30C.

Preparation of plant extracts


The fresh plant leaves were brought to the laboratory and washed
under running tap water; they were air dried and then homogenized
to fine powder and stored in airtight bottles. The dried powder of S.
sesban and R. communis was successively mixed with methanol for

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Afr. J. Microbiol. Res.

Biodeterioration of wall painting (Seti tomb)


Biodeterioration of wall painting (Seti tomb)
Figure 2. Biodeterioration of wall tomb painting.

Detailed view show fungal degradation (discoloration,


detachment
of plaster
pigments
Detailed view
show and
fungal
degradation (discoloration
,detachment of plaster and pigments

Table 1. Microbial inhibition zone (mm) of plant extracts concentration (mg/ml).

Test organism
F. oxysporum
R. stolonifer
A. flavus
S. warnei
M. luteus

Sesbania extract concentration (mg/ml)


20 mg
40 mg
80 mg
100 mg
9
12
13
13
10
10
12
13
8
9
11
12
8
9
12
14
10
11
12
15

72 h. The extracts were dried under reduced pressure using rotator


evaporator to get the crude. A dark green semi-solid mass was
obtained. It was stored below 4C until further used. The alcohol
extract was prepared by adding 10 g of crushed leaves of sesbania
or ricinus in 50 ml of methanol separately. It was allowed to stand
for 24 h after which it was filtered using a Whatman No. 1 filter
paper. The filtrate was directly used as crude extract with 100%
concentration. Further dilutions were made by adding appropriate
amount of methanol.

Microbial culture and media


The microbial cultures were prepared in microbiology agriculture lab
and used in the present study. The bacteria rejuvenated in nutrient
broth at 37C for 18 h and then stocked at 4C in nutrient agar
(Khante et al., 2008). Subcultures were prepared from the stock for
bioassay. The inoculum size of the bacterial culture was
standardized according to the National committee for Clinical
Laboratory Standards guideline. The bacterial culture was
inoculated into sterile nutrient broth and incubated at 37 oC. The
fungi medium is Potato Dextrose Agar PDA (200 g potato infusion,
20 g agar + 20 g Dextrose+1 L distilled water) and the bacterial
medium is nutrient agar NA (0.5% peptone, 0.3% beef extract/yeast
extract, 1.5% agar, 0.5% NaCl, 1 L distilled water).

Preparation of disc for antimicrobial activities


The sterile blotting paper disc (5 mm) was soaked in the diluted
extract in different concentrations (20, 40, 80 and 100%). The
prepared disc was dried in controlled temperature to remove excess

Ricinus extracts concentration (mg/ml)


20 mg
40 mg
80 mg
100 mg
8
10
13
13
7
10
11
12
9
12
12
13
8
12
12
14
10
11
12
13

of solvent. The modified paper disc diffusion (Delignette-Muller and


Flandrois, 1994) was employed to determine the antimicrobial
activity of aqueous extract of the herbal preparations. Inoculum was
spread over the agar plate using a sterile cotton swab in order to
obtain uniform microbial growth. Then the prepared antimicrobial
discs were kept over the lawn and pressed slightly along with
control. Streptomycin 10 g/disc was used as positive control. The
plates were incubated for 48 h at 37C. The antimicrobial activity
was evaluated and diameter of inhibition zones was measured.
Experiment was carried out in triplicate and the averages diameter
of zone of inhibition was recorded. The antibacterial activity was
classified as highly active (>10 mm), mildly active (7-10 mm) and
slightly active (6-7 mm); and less than 6 mm was taken as inactive
(Chandra et al., 2011).

RESULTS AND DISCUSSION


Effect of plant extracts on microbial strains
The antimicrobial activity of methanol extracts of S.
sesban stem portion was studied against Staphylococcus
warnei and Micrococcus luteus by zone of inhibition
method. The dried leaves of S. grandiflora are used in
some countries as tea which is considered to have
antibiotic properties (Gupta et al., 2008). The zone of
Inhibition study results are depicted in Table 1. The result
indicates that after 48 h methanol extracts (100 mg/ml)
showed zone of inhibition of 15 mm against M. luteus,
and 14 mm against S. warnei. The fungal group zone of

Khalaphallah and El-Derby

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Figure 3. Effect of different sesbania concentration on microbial inhibition zone.

Figure 4. Effect of different Ricinus concentration on microbial inhibition zone.

inhibition (100 mg/ml), after 72 h, was 12.3 mm against


Fusarium oxysporum; 12.1 mm against Rhizopus
stolonifer and 12.6 mm against Aspergillus flavus. The
graphical representation of zone of inhibition vs different
concentration of plant extracts after different time
intervals (48 h for bacteria and 72 h for fungi) are shown
in Figures 3 and 4 respectively.
According to Sandeep et al. (2014), cloroform and
methanol extracts of stem of S. sesban (25 mg/ml)
equally inhibit P. aeruginosa after 48 h. Both chloroform
and methanol extracts at a concentration of 100 mg/ml
were more effective against B. subtilis than other two
bacterial strains after 48 h. No antibacterial activity was
observed for pet ether ethanol and water extracts in a
range of 25 to 100 mg/ml. The methanol leaf extract of R.
communis exhibited maximum antimicrobial activity, and
water extract showed minimum activity against four
bacteria (S. aureus, B. subtilis, P. aeruginosa and K.
pneumonia) and two fungal strains (A. fumigatus and A.
flavus). These results are in agreement with the previous

work which showed that in plants most of the compounds


having antimicrobial potential are soluble in methanol
(Chandrasekaran et al., 2004), and low activity of water
extract is also reported by Ashafa et al. (2008).
R. communis showed good activity against M. luteus,
S. warnei, F. oxysporum, R. stolonifer and A. flavus. The
antimicrobial assay revealed that the methanol extracts of
leaves of R. communis possess good zone of inhibition,
whereas alcohol extract has antimicrobial activity only in
higher concentration (Table 1). Significant susceptibility
was recorded by most of the organisms tested with
methanol extract of leaves of R. communis, which
showed a comparatively reduced susceptibility pattern
(Figure 5).
The susceptibility pattern exhibited by the tested
organisms to these extracts could be exploited for
probably medicinal purposes in chemotherapy among
humans. With the current spread of antibiotic resistance
almost at geometric scale (Olayinka et al., 2004), proper
attention should be given to such plants to reap the

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Afr. J. Microbiol. Res.

Figure 5. Inhibition zone of antibacterial activity of TiO2 nanoparticles and plant


extracts at different.

Inhibition zone (MM)

Figure 6. Inhibition zone of antifungal activity of TiO2 nanoparticles and plant extracts at different
concentrations.

Figure 7. Effect of different nano TiO2 concentration on microbial inhibition zone.

potential antimicrobial benefits inherent in them.


However, actual antimicrobial ingredients need to be
extracted and identified; also its tolerable levels in the
human body as well as any toxic effects on human and
animal tissues need to be investigated accordingly.

Effect of Nano-TiO2 on microbial growth


The effect of chemical synthesized TiO2 nanoparticles on
the growth of microbial strains is shown in Figures 6 and
7. The antimicrobial activity is probably derived, through

Khalaphallah and El-Derby

Table 2. Microbial inhibition zone (mm) of Nano TiO2


concentration (g/ml).

Test organisms
F. oxysporum
R. stolonifer
A.
flavus
S. warnei
M. luteus

20 g
8
7
9
8
8

40 g
11
9
9
12
10

80 g
12
11
11
13
11

160 g
13
11
12
14
13

the electrostatic attraction between negatively charged


cell membrane of microorganism and positively charged
nanoparticles (Hamouda et al., 2001; Dibrov et al.,
2002; Dragieva et al., 1999), interaction of metal ions
including titanium with microbes (Amro et al., 2000) and
orientation of TiO2 (Wang et al., 2007).
Chemical TiO2 treatments had significant inhibitory
effect on the growth of microbes during 24 and 72 h of
incubation. The growth of the medium was investigated
as the number of microbes after contact with nanoparticles. The growth inhibition of the microbe by both
treatments recorded as a function of time suggested
significant differences in antimicrobial activity of the
nanoparticles (Table 2).
Chemically synthesized TiO2 NPs has a stronger
inhibitory effect on microorganisms. TiO2 NPs at a
concentration 20, 40, 80 and 160 inhibited growth of
bacterial and fungal strains, whereas the effect was much
less at lower concentration (Kon and Rai, 2013). The
efficacy of antimicrobial in terms of zones of inhibition
(mm) was measured against five different fungal and
bacterial strains (Table 2). The decreasing order of the
average antimicrobial activity of NPs against fungal group
was observed to be F. oxysorum A.flavus R.
stolonifer. The average antimicrobial activity of the
various NPs concentrations against bacterial group and
fungal group inhibition zone ranged from 814 and 8-13
mm, respectively. Increasing concentration of TiO2
nanoparticle decreases the growth of microbes, and the
concentration at which growth stopped altogether was
high with TiO2 chemically synthesized nanoparticle.
According to Adams et al. (2006), nanoparticles inhibited
growth of gram-positive by 90% but gram-negative was
much more resistant.
The antimicrobial ability of nano-TiO2 might be referred
to their small size which is 250 times smaller than a
bacterium. This makes them easier to adhere with the
cell wall of the microorganisms causing its destruction
and death of the cell. Also, metal nano-particles are
harmful to bacteria and fungi (Chwalibog et al., 2010).
Nano-TiO2 stimulates biofilm production and aggregate
within this bio-film. They bind closely to the surface of
microorganisms causing visible damage to the cells, and
demonstrating good self-assembling ability. Nano-TiO2
possesses well-developed surface chemistry, chemical
stability which makes them easier to interact with the

1429

microorganisms (Nirmala and Pandian, 2007). The


particles interact with the building elements of the outer
membrane and might cause structural changes, degradation and finally cell death. The effectiveness of TiO 2
can be explained on the basis of the oxygen species
released on the surface of TiO2, which cause fatal
damage to microorganisms (Sunada et al., 1998). The

generation of highly reactive species such as OH ,


H2O2 andO2 is explained as follows. Since TiO2 with
defects can be activated by both UV and visible light,
electron-hole pairs (eh+) can be created. The holes split
H2O molecules (from the suspension of TiO2) into

+
OH and H . Dissolved oxygen molecules are transformed
to superoxide radical anions (O2), which in turn react
with H+ to generate (HO2) radicals, which upon
subsequent collision with electrons produce hydrogen
peroxide anions (HO2). They then react with hydrogen
ions to produce molecules of H2O2. The generated
H2O2 can penetrate the cell membrane and kill the
bacteria (Fang et al., 2006). Since the hydroxyl radicals
and superoxide are negatively charged particles, they
cannot penetrate into the cell membrane and must
remain in direct contact with the outer surface of the
bacteria cell of the bacteria; however, H2O2 can penetrate
into the cell (Blake et al., 1999). The mechanisms of TiO 2
nanoparticles depend on the bactericidal effect which is a
result of mechanical interactions on cell walls and/or
membranes of microorganisms in the presence of
photocatalysis.
Conclusion
In this work we have reported the evidence of toxic
effects of Titanium dioxide nanoparticles (TiO2 NPs) and
plant extracts on different pollution microorganisms. The
antimicrobial activity of ethanol extracts was evaluated by
disc diffusion plate method. All the extracts were
evaluated in concentration range from 20 to 100 mg/ml.
Wall painting in the royal tombs (Tausert - Setnkht and
Seti I) suffers from microbial enzymes degradation and
needs urgent treatment. The lab test was performed by
using various concentrations of TiO2 NPs; 160 ppm was
the efficient concentration level in eliminating microbial
growth. TiO2 NPs and plant extracts did not cause any
observable alterations or color changes to pigments and
binding medium (Arabic gum) used in wall paintings. The
order of bacterial strains inhibition of methanol extract
after 48 h is as follows: S.warnei >M. luteus>
F.oxysporium> A. flavus > R. stolonifer. So, it is
concluded that Sesbania and Ricinus extracts possess
good antibacterial activity. The antibacterial activity may
be due to the presence of flavonoids and phenolic
compounds.
Conflict of interests
The authors did not declare any conflict of interest.

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Afr. J. Microbiol. Res.

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